Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is responsible for huge global suffering. Mtb is highly adaptive being able to survive in a wide variety of microenvironments as well as persist for decades in the human host as a latent infection. Growing resistance to first, second and third line drugs are driving a need for new treatments. To combat drug-resistance and persistent infections, we need to develop effective compounds against non-replicating Mtb. Targeting protein secretion mechanisms essential for bacterial survival is one way to meet this need. Protein secretion is a key cellular process which underpins bacterial survival, promoting virulence and allowing Mtb to detect and respond to its environment. The signal peptidase, LepB, plays an integral role in this process and is essential for survival; however few studies have been conducted to characterize LepB activity and explore its role in different physiological states. In this application we propos to develop and/or run high-throughput screening (HTS) assays to identify chemical inhibitors of the Mtb signal peptidase, LepB. We think that by inhibiting this gatekeeper protein, we will inhibit a large number of pathways critical for bacterial survival and persistence. We will take an innovative, target-driven, cell-based approach, as well as a traditional biochemical approach, to develop high throughput assays and screen for small molecule inhibitors of LepB. We will conduct screens and combine these with other assays to prioritize compounds. Small molecules identified from this work will be used as probes better to understand the physiology of tuberculosis, the changes in the secretome related to persistence and virulence, and as pre-therapeutic drug discovery agents. Because these early drug discovery compounds will act through a unique mechanism of action, they will likely be effective against multi-drug resistant and extremely multi-drug resistant TB. The significance of our proposal is that it has a dual outcome; regeneration of the early drug pipeline by enabling HTS and stimulation of basic biological studies by providing chemical probes.